The present invention relates to novel cytochrome P-450 enzymes.
The enzyme cytochrome P-450 has been found in animals, plants, and microorganisms. The cytochrome P450-dependent monooxygenase system catalyses the biosynthesis of important physiological compounds, and participates in the metabolism of foreign substances, xenobiotics. It can use as a substrate a wide range of natural products and drugs.
For its enzymatic action, cytochrome P-450 requires a coenzyme such as MAD(P)H, an electron-transport protein such as ferredoxin, and molecular oxygen. The following mechanism is postulated for metabolism of a substrate S to give a product SOH, with Fe indicating the active site of the cytochrome P-450: ##STR1##
In this mechanism, the electrons which effect reduction are provided from AND(P)H through the mediating effect of the electron-transport protein.
In general, cytochrome P-450 enzymes have mainly been isolated from eucaryotes, and are insoluble in water.
Cytochrome P-450 enzymes isolated from procaryotes are also known, including P-450.sub.cam from Pseudomonas putida [J Biol Chem (1974) 249, 94]; P-450.sub.BM-1 and P-450.sub.BM-3 both from Bacillus megaterium ATCC 14581 [reported respectively in Biochim Hiophys Acta (1985) 838, 302 and J Biol Chem (1986) 261, 1986, 7160]; P-450a, P-450b, and P-450c from Rhizobium japonicum [Biochim Biophys Acta (1967) 147, 399]: and P-450.sub.npd from Nocardia NHI [Microbios (1974) 9, 119].
Cytochrome P-450 enzymes purified from Streptomyces microorganisms remain relatively unreported. The induction of a cytochrome P-450 in Streptomyces griseus by soybean flour is described in Biochem and Biophys Res Comm (1986) 141, 405. The isolation and properties of two forms of a 6-deoxyerythronolide B hydroxylase from Saccharopolyspora erythraea (originally classified as Streptomyces erythraeus) is described in Biochemistry (1987) 26, 6204.
In European Patent Specification 215,665 published on Mar. 25, 1987 and in corresponding U.S. Ser. No. 07/393,001, Aug. 11, 1989, which is a continuation of U.S. Ser. No. 07/203/649, Jun. 1, 1988, which is a continuation of U.S. Ser. No. 06/906,034 filed Sep. 10, 1986, there is described an enzymatic hydroxylation. Hydroxylation is effected with a hydroxylation enzyme produced by a microorganism of the genus Streptomyces or of the genus Nocardia.
Four newly isolated strains of Streptomyces which produce suitable hydroxylation enzymes are described in EP 215,665 and U.S. Ser. No. 07/393,001, which is a continuation of U.S. Ser. No. 07/203,649, which is a continuation of U.S. Ser. No. 06/906,034, including Streptomyces sp SANK 62585.
Streptomyces sp SANK 62585 was deposited with the Fermentation Research Institute, Japan, on Sep. 5, 1985 under the accession number FERM P-8440, and re-deposited under the Budapest Treaty on Aug. 13, 1986 under the accession number FERM BP-1145.